Autonomic Nerves Regulate Gene Expression Profiles in The Developing Ductus Arteriosus

Abstract

Background The ductus arteriosus (DA), a bypass vessel between the aorta and main pulmonary artery, is essential for fetal circulation, and immediately closes after birth. Prostaglandin E2 plays important roles in both DA patency and closure via its receptor EP4. During the fetal periods, EP4 signaling dilates the DA through inhibition of myosin light chain kinase and promotes luminal narrowing, i.e., intimal thickening, for complete closure of the DA after birth when placental PGE2 is dramatically decreased. Previous studies have demonstrated that EP4 is prominently expressed in the developing DA and EP4-deficient mice exhibits patent DA after birth, suggesting that EP4 expression in the DA is critical for adaptation from fetal and adult circulatory system. However, the molecular mechanism of specific expression of EP4 in the DA is unknown. It has been reported that sympathetic nerves are distributed around the DA. Therefore, we hypothesized that sympathetic nerves regulated EP4 expression in the DA. In this study, we aimed to investigate the molecular mechanism of EP4 expression in the DA. Methods We generated EP4 reporter mice with EP4-IRES-nlsLacZ. EP4 reporter mice were stained with X-gal, and then tissue sections were prepared. We examined neurite outgrowth by immunohistochemistry with an antibody against polysialylated neural cell adhesion molecule (PSA-NCAM). Distribution of autonomic nerve terminals were detected by immunohistochemistry with an antibody against tyrosine hydroxylase. Results X-gal staining showed that no EP4 expression was observed in the DA at embryonic day 13 (E13). At E14, EP4 expression was observed in the smooth muscle layer of the DA, which was stronger in the outer layer (Figure 1). At E18.5 (full-term), EP4 expression level was significantly higher than pre-term mice and EP4 was uniformly expressed throughout the tunica media of the DA. At postnatal day 2, EP4 expression was decreased throughout the smooth muscle layer of the DA. No visible EP4 expression was observed in the smooth muscle layers of adjacent arteries. i.e., the aorta and pulmonary arteries, and endothelial cells of the DA and adjacent arteries. PSA-NCAM-positive neurites invaded into the adventitia toward the smooth muscle layer of the DA at E13, in which EP4 was not yet expressed. Neurite outgrowth became prominent in the tunica media in the DA at E14 (Figure 2). At full-term, neurites were observed throughout the smooth muscle layer of the DA, which was consistent with the area of the EP4 expression. Neurite outgrowth into the tunica media was not observed in the adjacent arteries. Furthermore, tyrosine hydroxylase was observed in the outer layer of the tunica media and the adventitia in the DA, indicating the presence of autonomic nerve terminals in the DA. Conclusion Sympathetic nerves may regulate specific expression of EP4 in the DA. These data suggest the novel epigenetic regulation of autonomic nerve-mediated vascular development.